Indolethylamine N-methyltransferase (INMT) is a transmethylation enzyme that utilizes the methyl donor S-adenosyl-L-methionine to transfer methyl groups to amino groups of small molecule acceptor compounds. INMT is best known for its role in the biosynthesis of N,N-Dimethyltryptamine (DMT), a psychedelic compound found in mammalian brain and other tissues. In mammals, biosynthesis of DMT is thought to occur via the double methylation of tryptamine, where INMT frst catalyzes the biosynthesis of N-methyltryptamine (NMT) and then DMT. However, it is unknown whether INMT is necessary for the biosynthesis of endogenous DMT. To test this, we generated a novel INMTknockout rat model and studied tryptamine methylation using radiometric enzyme assays, thin-layer chromatography, and ultra-high-performance liquid chromatography tandem mass spectrometry.We also studied tryptamine methylation in recombinant rat, rabbit, and human INMT. We report that brain and lung tissues from both wild type and INMT-knockout rats show equal levels of tryptaminedependent activity, but that the enzymatic products are neither NMT nor DMT. In addition, rat INMT was not sufcient for NMT or DMT biosynthesis. These results suggest an alternative enzymatic pathway for DMT biosynthesis in rats. This work motivates the investigation of novel pathways for endogenous DMT biosynthesis in mammals.